Insights into the Functioning of Bacillus subtilis HPr Kinase/Phosphatase: Affinity for Its Protein Substrates and Role of Cations and Phosphate
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- 作者：Jean-Pierre Lavergne ; Jean-Michel Jault ; and Anne Galinier
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：May 21, 2002
- 年：2002
- 卷：41
- 期：20
- 页码：6218 - 6225
- 全文大小：119K
- 年卷期：v.41,no.20(May 21, 2002)
- ISSN：1520-4995
文摘
In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase(HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 ofeither HPr (histidine-containing protein) or Crh (catabolite repression HPr). By using a surface plasmonresonance approach, it was shown here that the presence of magnesium is a prerequisite for the interactionof HprK/P with either HPr or Crh. HprK/P binds both protein substrates with a similar affinity (KD ofabout 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate. Inaddition, the specificity and the concentration of the cation required for the binding of protein substratesare different from that exhibited by the cation-binding site involved in the nucleotide binding, suggestingthe presence of two cation-binding sites on HprK/P. The effects of phosphate on enzymatic activities ofHprK/P were also investigated. Phosphate was able to unmask the phosphatase activity, especially in thepresence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinaseactivity of HprK/P. An apparent competition between phosphate and a fluorescent analogue of nucleotideled to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of theenzyme.